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11043 Background: The management of breast cancer (BC) patients (pts) with germline p53 mutations, a rare genetic condition, is not the object of specific recommendations. Preclinical data and preliminary clinical observations suggest a theoretically major radio-sensitivity and high risk of secondary radio-induced malignancies. It remains discussed whether the knowledge of a germline p53 mutation may influence treatment (trt) choices and have prognostic importance. We reviewed our cohort of BC diagnosed as first tumor in pts with germline p53 mutations within the past 11 years, with an attempt to describe secondary malignancies occurring after trt of primary tumor. Patients and Methods: 9 pts have been diagnosed and treated in our institution within the past 11 years for BC as first tumor in the context of a documented germline p53 mutation. Their tumor characteristics, treatment and follow-up data were extracted from prospectively-registered medical records. Results: Median age at diagnosis of primary BC was 32 (22–48). 7/9 pts had a family history compatible with Li Fraumeni syndrome, while 2 had no family history. No pt had previous knowledge of her p53 mutation. Primary tumors were ductal carcinoma in situ (4), infiltrating ductal carcinoma (4), phyllode tumor (1). 3 pts had conservative surgery and 6/9 underwent mastectomy (M). 6 received loco-regional radiation therapy (RT). None had prophylactic contra lateral mastectomy. Loco-regional (LRR) and contra-lateral relapses are listed in the Table , as well as incidence of second primaries within or outside radiation field. Second primaries were sarcoma in 3/4 cases and 1 was papillary thyroid carcinoma. Conclusions: Because of high risk of second breast primary and probably very high risk of radio-induced breast cancer, BC pts with germline p53 mutations should be advised bilateral prophylactic mastectomy and avoidance of radiation therapy. In this context, the knowledge of the mutation might be of great importance. [Table: see text] No significant financial relationships to disclose.

Introduction Le cetuximab est un anticorps chimerique monoclonal de type IgG1 dirige contre le recepteur du facteur de croissance epidermique (EGFR). Il est efficace dans les cancers colorectaux metastatiques (CCRm) refractaires a l’irinotecan et non mutes pour KRAS. Le cetuximab pourrait aussi exercer son activite antitumorale par des mecanismes de type cytotoxicite cellulaire dependante des anticorps (ADCC). Ce phenomene peut induire la mort des cellules tumorales par interaction entre le fragment Fc de l’anticorps lie a la cellule tumorale et les recepteurs Fc des cellules effectrices du systeme immunitaire. Les recepteurs FcγRIIa et FcγRIIIa presentent des polymorphismes constitutionnels (FcγRIIa-H131R et FcγRIIIa-V158F) qui pourraient affecter leur affinite avec les IgG1. De tels polymorphismes ont un impact sur la reponse et la survie de patients atteints de lymphome folliculaire et de cancer du sein metastatique, respectivement traites par rituximab et trastuzumab, anticorps therapeutiques egalement de type IgG1. Le but de l’etude etait de correler le statut KRAS et les polymorphismes de FcγRIIa et FcγRIIIa au taux de reponse et a la survie sans progression (SSP) des patients atteints de CCRm traites par cetuximab et irinotecan, initialement refractaires a l’irinotecan. Patients et Methodes Les ADN tumoraux et normaux, extraits a partir de materiel fixe et inclus en paraffine de 69 patients atteints de CCRm, ont respectivement fait l’objet de recherche de mutations de KRAS par technique SNaPshot apres PCR de l’exon 2 et des polymorphismes des recepteurs FcγRIIa et FcγRIIIa par sequencage direct et PCR multiplex. Resultats Le statut KRAS mute, detecte chez 39 % des patients, etait associe avec un plus faible taux de reponse (4 % chez les patients KRAS mute vs 27 % chez les patients KRAS non mute, p = 0,021) et a une SSP plus courte (3 νs 5,3 mois, p = 0,021). Les patients avec les genotypes FcγRIIa-131HH et/ ou FcγRIIIa-158VV (32 % des cas) avaient une SSP plus longue que ceux qui presentaient les deux alleles 131R et 158F (5,5 νs 3 mois, p = 0,005). Cette difference restait significative chez les patients mutes pour KRAS. En analyse multivariee, le statut KRAS et la combinaison des genotypes constituaient des facteurs independants pour la SSP. L’analyse combinee de ces parametres montrait que le taux de reponse et la SSP augmentaient avec le nombre de facteurs favorables (KRAS non mute et genotypes FcγRIIa-131HH et/ou FcγRIIIa-158VV). Conclusion Notre etude confirme la valeur predictive du statut KRAS chez les patients atteints de CCRm traites par cetuximab et irinotecan. Elle montre un impact favorable de la combinaison des genotypes FcγRIIa-131HH et/ou FcγRIIIa-158VV sur la SSP dans la meme population de patients. Un role important de l’ADCC dans l’activite antitumorale du cetuximab peut etre suggere notamment parce que ces polymorphisms restent favorables dans le groupe de CCRm mutes pour KRAS, ou la voie de l’EGFR est constitutivement activee. Les polymorphismes des recepteurs FcγRIIa / FcγRIIIa pourrait ainsi s’integrer a de nouveaux facteurs predictifs.

Introduction Les mutations de KRAS ont ete identifiees comme marqueurs de resistance aux anticorps monoclonaux anti-EGFR ( Epidermal Growth Factor Receptor ) dans le cancer colorectal metastatique (CCRM). Par ailleurs, l’efficacite de ces anticorps necessite que l’activation de la voie de l’EGFR contribue a la transformation maligne. Des etudes recentes suggerent que p53 agit comme un frein sur la voie de l’EGFR et que cette voie ne contribue a la carcinogenese que si p53 est inactivee. Cette observation suggere ainsi que les tumeurs comportant une mutation de TP53 seraient plus sensibles aux anticorps anti-EGFR. A notre connaissance, cette hypothese n’a jamais ete evaluee dans le CCRM [1, 2]. But Nous avons evalue l’impact des mutations somatiques de TP53 en combinaison avec les mutations de KRAS sur l’evolution clinique des patients traites par une chimiotherapie a base de cetuximab pour un CCRM. Materiels et Methodes Les tumeurs de 64 patients ont ete etudiees. Les mutations ont ete recherchees par sequencage direct des exons 5 a 8 pour TP53 et par la technique du SnaPshot pour KRAS. La reponse clinique a ete correlee aux statuts mutationnels par le test de Fisher. Le controle de la maladie etait defini par une reponse complete, partielle ou une stabilite. Les survies sans progression ont ete calculees selon la methode de Kaplan Meier et comparees par le test du log rank. Les facteurs predictifs cliniques et biologiques de survie sans progression ont ete recherches par une analyse multivariee selon le modele de Cox. Resultats Une mutation de KRAS a ete retrouvee chez 18 patients (28 %). Ces mutations etaient significativement associees a une progression de la maladie (p = 0,044). La survie sans progression des patients mutes pour KRAS etait significativement inferieure a celle des patients non mutes (12 vs 20 semaines, p = 0,034). Une mutation de TP53 a ete retrouvee chez 29/39 (74 %) patients avec une maladie controlee et chez 12/25 (48 %) patients avec une maladie en progression. Sur la population totale de l’etude, les mutations de TP53 etaient significativement associees au controle de la maladie (p = 0,037), et la survie sans progression etait significativement augmentee chez les patients presentant une mutation de TP53 (20 νs 12 semaines, p = 0,004). Chez les 46 patients sans mutation de KRAS, les mutations de TP53 etaient egalement significativement associees au controle de la maladie (p = 0,008) et la survie sans progression des patients mutes pour TP53 etait significativement superieure a celle des patients non mutes (24 νs 12 semaines, p = 0,0007). En analyse multivariee, les mutations de TP53 et KRAS ont ete identifiees comme les deux seuls facteurs independants predictifs de survie sans progression (HR = 1,99, IC : 1,09 - 3,63, p = 0,024 et HR = 0,48, IC : 0,25 - 0,94, p = 0,032 respectivement). Conclusion Notre etude suggere que chez les patients presentant un CCRM, les mutations de TP53 sont predictives d’une plus grande sensibilite au cetuximab, et en particulier en l’absence de mutation de KRAS. Le genotypage de TP53 pourrait ainsi avoir une valeur additionnelle chez les patients sans mutation de KRAS pour optimiser la selection des patients susceptibles de beneficier des therapies ciblees anti-EGFR. Remerciements, financements, autres Ce travail a recu le soutien financier de l’Association pour la Recherche sur le Cancer.

11004 Background: Cetuximab, a chimeric monoclonal IgG1 targeting the Epidermal Growth Factor Receptor (EGFR), has demonstrated clinical efficacy in mCRC patients refractory to Irinotecan, mainly i...

10502 Background: In metastatic colorectal cancer (MCRC), no molecular predictive markers to cetuximab response have been yet established. The aim was to evaluate whether KRAS gene mutations, EGFR immunochemistery (IHC) and EGFR gene copy number correlate with response to cetuximab. Methods: 59 patients with MCRC treated by cetuximab between July 2004 and December 2005 were retrospectively included. Clinical data were collected and tumour response was evaluated according to RECIST criteria. EGFR IHC was performed using the Dako kit. The EGFR gene copy number was determined by FISH (Fluorescence in-Situ Hybridization). Detection of KRAS gene mutations on exon 2 was performed by sequencing of extracted paraffin-embedded DNA and then by 2 methods, SNaPshot and PCR-LCR, specifically developed to detect small fractions of mutated tumor cells. Response to cetuximab was studied according to clinical data, IHC, FISH and KRAS mutation analysis using the Fischer exact test. Predictive factors of response were determined by logistic regression. Skin reactions were collected but not considered for this analysis as regards the lack of accurate grading in a retrospective study. Times to progression (TTP) were calculated using the Kaplan-Meier method and compared with log-rank test. Results: 12 patients (20.3%) responded to cetuximab (2 patients with complete response and 10 patients with partial response), 19 (32.2%) had stable disease and 28 (47.5%) were in disease progression. A KRAS mutation was detected in 22/59 tumours and, in 6 cases, was missed by sequencing analysis but detected using the SNaPshot and PCR-LCR assays. No KRAS mutation was found in responders patients. KRAS mutation was associated with disease progression (p = 0.0005) and TTP was significantly decreased in mutated KRAS patients (3 vs 5.5 months, p = 0.015). There was no correlation between EGFR IHC and cetuximab response. No EGFR gene copy number increase was detected in responders patients. Predictive factors of cetuximab resistance were KRAS mutation (p=0.003; OR:0.10; 95IC:0.22–0.40) and age No significant financial relationships to disclose.

The presenilin 1 (PS1) gene, located on chromosome 14, is the major gene involved in the autosomal dominant forms of early onset Alzheimer's disease (AD). In order to estimate the frequency of de novo PS1 mutations, we have sequenced the PS1 open reading frame in 13 clinically diagnosed patients with no affected relatives, who had developed AD before the age of 50. In one case with onset at 37 years, we identified a missense mutation resulting in a methionine to lysine substitution at codon 139 of the PS1 gene. This substitution is the fourth identified at the same codon. This study, in agreement with previous reports, suggests that de novo PS1 mutations can occur but at a low frequency.

Background: Few germline BRCA2 rearrangements have been described compared with the large number of germline rearrangements reported in the BRCA1 gene. However, some BRCA2 rearrangements have been reported in families that included at least one case of male breast cancer. Objective: To estimate the contribution of large genomic rearrangements to the spectrum of BRCA2 defects. Methods: Quantitative multiplex PCR of short fluorescent fragments (QMPSF) was used to screen the BRCA2 gene for germline rearrangements in highly selected families. QMPSF was previously used to detect heterozygous deletions/duplications in many genes including BRCA1 and BRCA2 . Results: We selected a subgroup of 194 high risk families with four or more breast cancers with an average age at diagnosis of ⩽50 years, who were recruited through 14 genetic counselling centres in France and one centre in Switzerland. BRCA2 mutations were detected in 18.6% (36 index cases) and BRCA1 mutations in 12.4% (24 index cases) of these families. Of the 134 BRCA1/2 negative index cases in this subgroup, 120 were screened for large rearrangements of BRCA2 using QMPSF. Novel and distinct BRCA2 deletions were detected in three families and their boundaries were determined. We found that genomic rearrangements represent 7.7% (95% confidence interval 0% to 16%) of the BRCA2 mutation spectrum. Conclusion: The molecular diagnosis of breast cancer predisposition should include screening for BRCA2 rearrangements, at least in families with a high probability of BRCA2 defects.

In a previous report [Jacquet et al., 2005] we have shown that mild to moderate hyperprolinemia resulting from several alterations (either a complete deletion or missense mutations) of the proline dehydrogenase (PRODH) gene located on chromosome 22q11 is a risk factor for schizoaffective disorder but not for DSM3 R schizophrenia or bipolar disorder. We now report that hyperprolinemia is not associated with childhood onset schizophrenia (COS).

Hereditary Hemorrhagic Telangiectasia (HHT) is an autosomal dominant disease characterized by arteriovenous malformations and resulting from mutations in two major genes: ENG and ACVRL1. The aim of the present study was to estimate the prevalence of the mutations of ENG and ACVRL1 in HHT, based on the largest series of patients reported so far, recruited through a national network. We previously reported the first mutation screening of both genes, in French HHT patients, using heteroduplex analysis. This previous study, bringing 60 novel mutations, provided a significant improvement to the knowledge of molecular pathology in HHT. However, 32% (n=48) of the patients with a confirmed clinical diagnosis remained without mutation. In these patients, we performed an extensive molecular analysis that included the sequencing of the whole coding sequence, the search for large rearrangements, and screening of the potential 5′ regulatory regions. Additionally, due to the lack of large pedigrees suitable for linkage analysis, and since SMAD4 germline mutations have been reported in families with combined HHT and juvenile polyposis, we screened this gene and five other genes involved in the TGF-β/BMP pathway in the patients without mutation of ENG or ACVRL1. Only a novel SMAD1 non-conservative substitution was found in one patient, changing a poorly conserved methionine to an isoleucin. Twenty-three mutations were found in ACVRL1 and 8 in ENG (including a duplication of exons 4 to 8 and deletions of exons 1 to 3 and 9 to 14). Our results, combined with our previous data, increase the mutation rate to 88% (n=119/136) in French patients with a confirmed clinical diagnosis. Our results also emphasize the higher prevalence of large insertions/deletions in ENG and the predominance of ACVRL1 over ENG mutations. © 2006 Wiley-Liss, Inc.

Since the original reports of hyperprolinaemia by Scriver et al 1 and Shafer et al ,2 two types of this rare metabolic disorder have been biochemically characterised3: type I (MIM 239500) results from the defect of the enzyme proline dehydrogenase (oxidase), which ensures the conversion of proline into Δ-1-pyrroline-5-carboxylate (P5C), the first step in the conversion from proline to glutamate,4 and type II (MIM 239510) is the result of a defect of the P5C dehydrogenase/aldehyde dehydrogenase 4 enzyme and P5C is excreted in the urine.5 The phenotype of type II hyperprolinaemia is characterised by neurological manifestations including seizures and mental retardation.3,6,7 Although type I hyperprolinaemia was originally described in a kindred with a familial nephropathy,1,2 the renal disease was subsequently shown to be coincidental and type I hyperprolinaemia has been considered to be a benign disorder which can be asymptomatic.3 Nevertheless, two studies have reported severe neurological manifestations (mental retardation, epilepsy) in two male children with type I hyperprolinaemia.8,9 While mutations of the ALDH4A1 gene, located on chromosome 1p36, have been identified in families with type II hyperprolinaemia,10 the molecular basis of type I hyperprolinaemia has not been characterised until recently. We have recently identified in schizophrenic patients a heterozygous deletion and mutations of the PRODH gene, located on 22q11, which were associated with moderate hyperprolinaemia. We …

Alzheimer's disease, the most common cause of dementia in later life, is genetically heterogeneous. Mutations in three genes encoding the amyloid precursor protein ( APP ), presenilin 1 ( PSEN1 ), and presenilin 2 ( PSEN2 ) are responsible for autosomal dominant early onset cases. A few families have been described in which PSEN1 mutations, usually exon 9 deletions, cause progressive dementia associated with spastic paraparesis.1–5 We present a family in which another PSEN1 mutation causes disease that begins with spastic paraparesis and is associated with dementia that is not of the Alzheimer type. The index case first presented at the age of 54 with lower back pain and gait difficulties; he was unable to squat unaided and walked with caution. He complained of a memory deficit that he attributed to the Algerian war, 34 years ago. On examination, he had brisk reflexes in all four limbs and normal muscle tone. Blood cell counts, CSF glucose and protein levels, electromyography, and nerve conduction velocity were unremarkable. A lumbar CT scan and cervicothoracic MRI showed no signs of spinal compression. Brain MRI showed mild cortical atrophy. One year later, the patient had a bilateral extensor plantar reflex, with clonus of the patella and proximal muscle weakness in the lower limbs. There was no …

To the Editor: A genetic variation in the 3′-untranslated region of the prothrombin gene was recently linked to an increased risk for venous thrombosis. In 28 families selected for hereditary thrombophilia, 5 (18%) of the probands carried the G to A transition at nucleotide 20210 of the factor II

La maladie d'Alzheimer a un determinisme tres heterogene. Dans les rares cas, a debut precoce et a transmission autosomique dominante, trois genes sont impliques : le premier, localise sur le chromosome 21, code pour la proteine precurseur de l'amyloide, le deuxieme, sans doute le plus frequemment en cause, est situe sur le chromosome 14 et code pour la preseniline 1 ; le troisieme code pour la preseniline 2, et est localise sur le chromosome 1. L'existence d'un quatrieme locus contenant un gene majeur est vraisemblable. A cote de ces genes a transmission dominante, d'autres genes sont des facteurs de risque pour la maladie ; on a trouve une association forte entre la maladie d'Alzheimer et l'allele e4 du gene codant pour l'apolipoproteine E. Il est encore trop tot pour proposer un mecanisme pathogenique unique impliquant l'ensemble de ces genes. Cependant, il est probable que l'action de plusieurs d'entre eux s'integre dans le cadre de la cascade amyloidergique, aboutissant a la constitution des plaques seniles et des lesions et symptomes associes.

To establish if CA 19-9 could detect early pancreatic cancer, we measured its serum concentration in 866 patients admitted for benign diseases and observed for 2 years. All patients with an elevated CA 19-9 level (greater than 40 units (U)/ml) were submitted to a computed tomography (CT) scan of the pancreas. The CA 19-9 level was increased in 117 patients. One hundred fifteen of these 117 patients had false-positive elevations. The CA 19-9 concentration was elevated mostly in benign hepatobiliary diseases. In this group of patients, CA 19-9 was correlated to alkaline phosphatase values. Eleven patients showed an elevated CA 19-9 level for 10 months without any malignancy developing. One patient had a normal CA 19-9 concentration 8 months before clinical signs of pancreatic carcinoma developed. We conclude that CA 19-9 measurement is of no value for the early detection of this malignancy.

The diagnostic value of hyaluronate concentration in effusions of malignant mesothelioma has been extensively reported but no information is available about serum hyaluronate in patients with this cancer. Using a new enzymoimmunologic assay based on hyaluronate-hyaluronectin interaction, serum levels of hyaluronate were measured in 16 patients with malignant pleural mesothelioma, 50 patients with other pleural effusions, and 94 healthy blood donors. The mean serum hyaluronate level in patients with mesothelioma (mean, 750 micrograms/l; range, 29 to 5833 micrograms/l) was significantly higher than in patients with other pleural effusions (mean, 56 micrograms/l; range, 4 to 137 micrograms/l) and than in blood donors (mean, 24 micrograms/l; range, 0 to 94 micrograms/l). Comparison of serum hyaluronate values observed in mesotheliomas with the clinical course of the disease suggests that serum hyaluronate might increase only at an advanced stage of the cancer. Therefore, serum hyaluronate determination has probably no clinical value for early detection of malignant mesothelioma, but might be useful to evaluate the clinical course of this malignancy.

It has been suggested that glycosaminoglycans are involved in the pathogenesis of liver fibrosis. Furthermore, recent studies have reported that one of them, hyaluronate, was mainly taken up and degraded by the liver. Using an enzymoimmunological assay, based on hyaluronate-hyaluronectin interaction, serum levels of hyaluronate were measured in 113 patients with various liver diseases. Patients were divided into six groups according to clinical, biological and histological data: Group 1-alcoholic cirrhosis (n = 47) including alcoholic cirrhosis with alcoholic hepatitis (n = 24); Group 2-primary biliary cirrhosis (n = 21); Group 3-cirrhosis related to viral hepatitis (n = 10); Group 4-idiopathic hemochromatosis (n = 17); Group 5-alcoholic fatty liver (n = 8); and Group 6-viral or drug acute hepatitis (n = 10). Ninety-four blood donors were studied as controls. Levels of hyaluronate were found to be strikingly elevated in Group 1 (1,225 +/- 1,137 micrograms per liter), Group 2 (792 +/- 739 micrograms per liter), Group 3 (649 +/- 373 micrograms per liter), and Group 4 (246 +/- 242 micrograms per liter), whereas patients in Group 5 (94 +/- 63 micrograms per liter) and Group 6 (73 +/- 57 micrograms per liter) had values close to controls (23 +/- 17 micrograms per liter). There was a significant correlation between serum hyaluronate and serum albumin, prothrombin time, factor V concentration and serum gamma-globulins. It is suggested that hyaluronate levels reflect both active fibrosis and hepatic failure and may be a quantitative marker of severity of hepatic injury.

Fifty years after the recognition of the Li-Fraumeni syndrome (LFS), our perception of cancers related to germline alterations of TP53 has drastically changed: (i) germline TP53 alterations are often identified among children with cancers, in particular soft-tissue sarcomas, adrenocortical carcinomas, central nervous system tumours, or among adult females with early breast cancers, without familial history. This justifies the expansion of the LFS concept to a wider cancer predisposition syndrome designated heritable TP53-related cancer (hTP53rc) syndrome; (ii) the interpretation of germline TP53 variants remains challenging and should integrate epidemiological, phenotypical, bioinformatics prediction, and functional data; (iii) the penetrance of germline disease-causing TP53 variants is variable, depending both on the type of variant (dominant-negative variants being associated with a higher cancer risk) and on modifying factors; (iv) whole-body MRI (WBMRI) allows early detection of tumours in variant carriers and (v) in cancer patients with germline disease-causing TP53 variants, radiotherapy, and conventional genotoxic chemotherapy contribute to the development of subsequent primary tumours. It is critical to perform TP53 testing before the initiation of treatment in order to avoid in carriers, if possible, radiotherapy and genotoxic chemotherapies. In children, the recommendations are to perform clinical examination and abdominal ultrasound every 6 months, annual WBMRI and brain MRI from the first year of life, if the TP53 variant is known to be associated with childhood cancers. In adults, the surveillance should include every year clinical examination, WBMRI, breast MRI in females from 20 until 65 years and brain MRI until 50 years.

We have constructed two vectors, pGCAT-A and pGCAT-C, designed to facilitate the construction of recombinant plasmids containing the bacterial gene (cat) coding for chloramphenicol acetyltransferase (CAT) under the control of eukaryotic promoter and/or enhancer elements. The cat gene was inserted downstream from a multiple cloning site (MCS) region with eleven unique restriction sites. The MCS region is in opposite orientation in the two vectors. The CAT activity of control extracts from cells transfected with pGCAT-A or pGCAT-C is very low. Insertion of the viral SV40 early promoter into one of the sites of the MCS region of pGCAT-A or pGCAT-C results in a 30- to 400-fold stimulation of the CAT activity.

Summary Aim of the study To detect hereditary non-polyposis colorectal cancer (HNPCC) patients with a strategy combining clinical selection (patient age at onset of cancer less than 50 years or family history of HNPCC tumors) and microsatellite instability typing plus immunohistochemistry, leading to mismatch repair (MMR) germline mutation analysis. Methods Tumors were screened for microsatellite instability (MSI) and for hmlh1 and hmsh2 immunohistochemical expression. Germline mutation analysis was performed to search for MLH1 and MSH2 mutations in patients with MSI-High and MSI-Low tumors. Results 197 adenocarcinomas were studied: 164 patients were ≤ 50 years old, 33 were older than 50 years but had a family history of HNPCC tumors. Fifty tumors (25.4%) were MSI-High, 10 were MSI-Low (5.1%), and 130 were MS-Stable (66%). MSI typing was inconclusive in 7 (3.5%). Immunohistochemistry screening was performed on 165 tumors: sensitivity was 63.6%, specificity was 99%. Germline mutation analysis was performed in 33/60 MSI-High or Low tumors: 23 mutations were noted (70% of the tested patients). Conclusion This proposed strategy of determining microsatellite instability in young colorectal cancer patients or in patients with a family history of HNPCC tumors led to an increased frequency in the detection of MMR germline mutations.

International audience; BACKGROUND: Sarcomas are rare mesenchymal malignancies whose pathogenesis is poorly understood; both environmental and genetic risk factors could contribute to their aetiology.METHODS AND RESULTS: We performed whole-exome sequencing (WES) in a familial aggregation of three individuals affected with soft-tissue sarcoma (STS) without TP53 mutation (Li-Fraumeni-like, LFL) and found a shared pathogenic mutation in CDKN2A tumour suppressor gene. We searched for individuals with sarcoma among 474 melanoma-prone families with a CDKN2A-/+ genotype and for CDKN2A mutations in 190 TP53-negative LFL families where the index case was a sarcoma. Including the initial family, eight independent sarcoma cases carried a germline mutation in the CDKN2A/p16INK4A gene. In five out of seven formalin-fixed paraffin-embedded sarcomas, heterozygosity was lost at germline CDKN2A mutations sites demonstrating complete loss of function. As sarcomas are rare in CDKN2A/p16INK4A carriers, we searched in constitutional WES of nine carriers for potential modifying rare variants and identified three in platelet-derived growth factor receptor (PDGFRA) gene. Molecular modelling showed that two never-described variants could impact the PDGFRA extracellular domain structure.CONCLUSION: Germline mutations in CDKN2A/P16INK4A, a gene known to predispose to hereditary melanoma, pancreatic cancer and tobacco-related cancers, account also for a subset of hereditary sarcoma. In addition, we identified PDGFRA as a candidate modifier gene

International audience; CONTEXT: Results of comparative genomic hybridization studies have suggested that rare copy number variations (CNVs) at numerous loci are involved in the cause of mental retardation, autism spectrum disorders, and schizophrenia. OBJECTIVES: To provide an estimate of the collective frequency of a set of recurrent or overlapping CNVs in 3 different groups of cases compared with healthy control subjects and to assess whether each CNV is present in more than 1 clinical category. DESIGN: Case-control study. SETTING: Academic research. PARTICIPANTS: We investigated 28 candidate loci previously identified by comparative genomic hybridization studies for gene dosage alteration in 247 cases with mental retardation, in 260 cases with autism spectrum disorders, in 236 cases with schizophrenia or schizoaffective disorder, and in 236 controls. MAIN OUTCOME MEASURES: Collective and individual frequencies of the analyzed CNVs in cases compared with controls. RESULTS: Recurrent or overlapping CNVs were found in cases at 39.3% of the selected loci. The collective frequency of CNVs at these loci is significantly increased in cases with autism, in cases with schizophrenia, and in cases with mental retardation compared with controls (P < .001, P = .01, and P = .001, respectively, Fisher exact test). Individual significance (P = .02 without correction for multiple testing) was reached for the association between autism and a 350-kilobase deletion located at 22q11 and spanning the PRODH and DGCR6 genes. CONCLUSIONS: Weakly to moderately recurrent CNVs (transmitted or occurring de novo) seem to be causative or contributory factors for these diseases. Most of these CNVs (which contain genes involved in neurotransmission or in synapse formation and maintenance) are present in the 3 pathologic conditions (schizophrenia, autism, and mental retardation), supporting the existence of shared biologic pathways in these neurodevelopmental disorders.

Background: According to the international criteria for hereditary non-polyposis colorectal cancer (HNPCC) diagnostics, cancer patients with a family history or early onset of colorectal tumours showing high microsatellite instability (MSI-H) should receive genetic counselling and be offered testing for germline mutations in DNA repair genes, mainly MLH1 and MSH2. Recently, an oncogenic V600E hotspot mutation within BRAF, a kinase encoding gene from the RAS/RAF/MAPK pathway, has been found to be associated with sporadic MSI-H colon cancer, but its association with HNPCC remains to be further clarified.Methods: BRAF-V600E mutations were analysed by automatic sequencing in colorectal cancers from 206 sporadic cases with MSI-H and 111 HNPCC cases with known germline mutations in MLH1 and MSH2. In addition, 45 HNPCC cases showing abnormal immunostaining for MSH2 were also analysed.Results: The BRAF-V600E hotspot mutation was found in 40% (82/206) of the sporadic MSI-H tumours analysed but in none of the 111 tested HNPCC tumours or in the 45 cases showing abnormal MSH2 immunostaining.Conclusions: Detection of the V600E mutation in a colorectal MSI-H tumour argues against the presence of a germline mutation in either the MLH1 or MSH2 gene. Therefore, screening of these mismatch repair (MMR) genes can be avoided in cases positive for V600E if no other significant evidence, such as fulfilment of the strict Amsterdam criteria, suggests MMR associated HNPCC. In this context, mutation analysis of the BRAF hotspot is a reliable, fast, and low cost strategy which simplifies genetic testing for HNPCC.

ContextSignificant proportions of patients with hamartomatous polyposis or with hyperplastic/mixed polyposis remain without specific clinical and molecular diagnosis or present atypically. Assigning a syndromic diagnosis is important because it guides management, especially surveillance and prophylactic surgery.ObjectiveTo systematically classify patients with unexplained hamartomatous or hyperplastic/mixed polyposis by extensive molecular analysis in the context of central rereview of histopathology results.Design, Setting, and PatientsProspective, referral-based study of 49 unrelated patients from outside institutions (n = 28) and at a comprehensive cancer center (n = 21), conducted from May 2, 2002, until December 15, 2004. Germline analysis of PTEN, BMPR1A, STK11 (sequence, deletion), SMAD4, and ENG (sequence), specific exon screening of BRAF, MYH, and BHD, and rereview of polyp histology results were performed.Main Outcome MeasuresMolecular, clinical, and histopathological findings in patients with unexplained polyposis.ResultsOf the 49 patients, 11 (22%) had germline mutations. Of 14 patients with juvenile polyposis, 2 with early-onset disease had mutations in ENG, encoding endoglin, previously only associated with hereditary hemorrhagic telangiectasia; 1 had hemizygous deletion encompassing PTEN and BMPR1A; and 1 had an SMAD4 mutation. One individual previously classified with Peutz-Jeghers syndrome had a PTEN deletion. Among 9 individuals with an unknown hamartomatous polyposis, 4 had mutations in STK11 (1), BMPR1A (2), and SMAD4 (1). Of the 23 patients with hyperplastic/mixed polyposis, 2 had PTEN mutations. Substantial discrepancies in histopathology results were seen.ConclusionsSystematic molecular classification of 49 patients with unexplained hamartomatous or hyperplastic polyposis uncovered a potential novel susceptibility gene, ENG, for juvenile polyposis. Importantly, given the substantial proportion of patients found to have germline mutations, more extensive analysis of the known susceptibility genes is indicated. Rereview of histology results by a dedicated gastrointestinal pathologist should be considered routinely, as organ-specific surveillance rests on defining syndromic diagnosis.

Phenylketonuria (PKU) is an autosomal recessive disease due to deficiency of a hepatic enzyme, phenylalanine hydroxylase (PAH). The absence of PAH activity results in typical PKU while persistence of a residual enzyme activity gives rise to variant forms of the disease. We report here a 3-base pair in-frame deletion of the PAH gene (delta 194) in a mild variant, with markedly reduced affinity of the enzyme for phenylalanine (Km = 160 nM), and we provide functional evidence for responsibility of the deletion in the mutant phenotype. Since the deletion was located in the third exon of the gene, which presents no homology with other hydroxylases, we suggest that exon 3 is involved in the specificity of the enzyme for phenylalanine. Finally, since none of the 98 PKU patients tested were found to carry this particular deletion, our study suggests that this molecular event probably occurred recently on the background of a haplotype 2 gene in Portugal.

Background The EGFR 3′ untranslated region (UTR) harbors a polyadenine repeat which is polymorphic (A13/A14) and undergoes somatic deletions in microsatellite instability (MSI) colorectal cancer (CRC). These mutations could be oncogenic in colorectal tissue since they were shown to result into increased EGFR mRNA stability in CRC cell lines. Methods First, we determined in a case control study including 429 CRC patients corresponding to different groups selected or not on age of tumor onset and/or familial history and/or MSI, whether or not, the germline EGFR A13/A14 polymorphism constitutes a genetic risk factor for CRC; second, we investigated the frequency of somatic mutations of this repeat in 179 CRC and their impact on EGFR expression. Results No statistically significant difference in allelic frequencies of the EGFR polyA repeat polymorphism was observed between CRC patients and controls. Somatic mutations affecting the EGFR 3′UTR polyA tract were detected in 47/80 (58.8%) MSI CRC versus 0/99 microsatellite stable (MSS) tumors. Comparative analysis in 21 CRC samples of EGFR expression, between tumor and non malignant tissues, using two independent methods showed that somatic mutations of the EGFR polyA repeat did not result into an EGFR mRNA increase. Conclusion Germline and somatic genetic variations occurring within the EGFR 3′ UTR polyA tract have no impact on CRC genetic risk and EGFR expression, respectively. Genotyping of the EGFR polyA tract has no clinical utility to identify patients with a high risk for CRC or patients who could benefit from anti-EGFR antibodies.

L’analyse des gènes responsables de formes mendéliennes de CCR reste parfois négative chez des patients présentant un Cancer ColoRectal (CCR) de survenue très précoce, situation clinique pourtant suggestive d’un déterminisme génétique fort. Plusieurs études ont suggéré la contribution de variants rares affectant des gènes de réparation de l'ADN au déterminisme du CCR mais le manque de significativité statistique à l’échelle de l’exome limite l'interprétation de ces résultats. Afin de déterminer la contribution des variations génétiques rares affectant la réparation de l'ADN au CCR, nous avons séquencé par Next Generation Sequencing (NGS) un panel de 201 gènes impliqués dans le cancer chez 84 patients atteints de CCR très précoce d'origine française rigoureusement sélectionnés (CCR diagnostiqué jusqu’à l'âge de 32 ans, exclusion du syndrome de Lynch et des polyposes adénomateuses et hamartomateuses). Nous avons également séquencé l’exome de 93 patients, sélectionnés selon les mêmes critères, et de 567 témoins FREX (French Exome Project). Nous avons filtré les variants rares prédits pathogènes, affectant les 201 gènes du cancer ou les 296 gènes de réparation de l'ADN selon l’analyse réalisée, et nous avons comparé leurs fréquences relatives entre les cas et les témoins. À l’aide d’un test statistique de charge en variants (Burden test SeqMeta), nous avons obtenu un Odds Ratio (OR) de 1,412 (p = 0,165) pour les variations Loss of Function (LOF) rares (Minor Allele Frequency, MAF < 1%) affectant les gènes de la réparation de l’ADN, et un OR de 1,681 (p = 0,017) pour les variations LOF très rares (MAF < 1‰). L’effet observé est plus fort lorsque le test est restreint aux variants très rares affectant le système de réparation des liaisons inter-brins (Inter-strand CrossLink, ICL), avec un OR de 5,88 (p = 0,012). Fait remarquable, la comparaison de la cooccurrence d'au moins deux variations LOF très rares affectant la réparation de l'ADN entre patients et témoins donne un OR de 7,196 (p = 0,077). Ces résultats posent la question de la contribution de la voie ICL au CCR et de la place de l’oligogénisme, basé sur l'effet cumulatif de plusieurs variations affectant en particulier la réparation de l'ADN, dans la prédisposition au CCR.

Le dermatofibrosarcome de Darier et Ferrand est un sarcome des tissus mous, généralement de bas grade de malignité et d'évolution lente, développé à partir du derme. Il s'agit d'un cancer rare avec une prévalence dans la population générale estimée entre 0,8 et 5 par million. Dans plus de 95% des cas, sont observées deux anomalies génétiques récurrentes correspondant soit à une translocation réciproque des bras longs des chromosomes 17 et 22, soit à un anneau chromosomique surnuméraire. Ces anomalies génétiques somatiques aboutissent toutes les deux à la fusion du gène COL1A1 et du proto-oncogène PDGFB. Ce gène de fusion a pour conséquence une hyperproduction de PDGFB responsable de la transformation maligne des cellules possédant le récepteur adapté : le PDGFRB. Ce récepteur à activité tyrosine kinase est impliqué dans plusieurs autres types de cancers et il constitue une cible pour les thérapies ciblées comme l'imatinib. Dans le cadre du diagnostic moléculaire du syndrome de Li-Fraumeni, nous disposons d'un recrutement national exceptionnel de familles présentant une agrégation de cancers rares. Nous avons ainsi noté la survenue dans certaines familles de plusieurs cas de dermatofibrosarcome ou son agrégation avec de multiples cas de cancers. Cette récurrence de cancers rares est évocatrice d'un risque génétique se transmettant selon un mode Mendélien. Nous avons testé, au cours de cette thèse d'exercice de médecine, l'implication des gènes PDGFB et PDGFRB dans le déterminisme Mendélien du dermatofibrosarcome de Darier et Ferrand. Nous avons choisi d'étudier ces deux gènes en raison de leur implication dans de nombreux types de cancers sporadiques. En effet, des mutations somatiques du gène PDGFB sont impliquées dans plus de 95% des dermatofibrosarcomes. De plus, une thérapie ciblée serait envisageable pour les cancers auxquels prédisposent ces deux proto-oncogènes. Nous avons donc recherché des mutations sur la totalité de la séquence codante du gène PDGFRB par PCR et séquençage. Puis, nous avons recherché des réarrangements de grande taille de ces deux gènes par QMPSF. Ainsi, cette étude n'a pas permis d'identifier les bases moléculaires des dermatofibrosarcomes et des nombreux cancers retrouvés dans ces familles. Il sera nécessaire de poursuivre cette étude par le séquençage du gène PDGFB et par la recherche d'une instabilité chromosomique dans ces familles.